Linear 25 kda polyethyleneimine pei

FlpInCHO cells were purchased from Thermo Fisher Scientific. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, hygromycin B, and D-luciferin were obtained from Life Technologies (Carlsbad, CA, USA). Coelenterazine h was purchased from Prolume (Pinetop, AZ, USA). Enzymes and other materials for molecular cloning were sourced from New England Biolabs (Ipswich, MA, USA). Linear 25 kDa polyethyleneimine (PEI) was from Polysciences (Warrington, PA, USA). The human chemokines CCL17, CCL19, CCL21, CCL22, CCL27 and CCL28 (1-108) were supplied by Peprotech (Cranbury, NJ, USA) and human CCL28(4-108) was from BioLegend (San Diego, CA, USA). White 96-well CulturPlates were purchased from PerkinElmer (Boston, MA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

All reagents, unless specified, were purchased from Fisher Scientific (Ottawa, ON). Dulbecco’s modified Eagle’s medium, fetal bovine serum and penicillin-streptomycin were purchased from Wisent (St-Bruno, Qc, Canada). Linear 25 kDa polyethyleneimine (PEI) was from Polysciences Inc. (Warrington, PA). Coelenterazine 400a and coelenterazine h were purchased from Prolume Ltd. (Pinetop, AZ). Brefeldin A was purchased from Molecular Probes (Eugene, OR). The protease inhibitor cocktail was purchased from Sigma-Aldrich (Oakville, ON). The following mouse monoclonal antibodies were used: anti-hemagglutinin (anti-HA) antibody (MMS-101P, Cedarlane Laboratories, Burlington, ON); anti-myc antibody (MMS-105P, Cedarlane Laboratories); anti-γ antibody (A4200, Sigma-Aldrich, Oakville, ON); anti-actin (612657, BD Biosciences, Mississauga, ON); anti-Renilla luciferase (MAB4410) antibody (16932-1-AP) was purchased from EMD Millipore (Billerica, MA). The following rabbit polyclonal antibodies were used: anti-GFP antibody (ab290, Abcam, Cambridge, MA); anti-ARF1 antibody (20226-1-AP, Proteintech, Chicago, IL). Anti-mouse and anti-rabbit horseradish peroxidase-conjugated IgG were from GE Healthcare (Chalfont St.Giles, Buckinghamshire, UK).

For the transient overexpression of PIWIL1, HCT 116 cells were transfected with the human full-length cDNA clone pCMV6-PIWIL1 (RC205269) or with control pCMV6-Entry mammalian vector (PS100001) (Origene, Herford, Germany). At 24 h prior to transfection, HCT 116 cells at the exponential growth phase were seeded in 100 mm culture dishes; the next day, plates at 60% confluency were washed and re-fed with culture medium shortly before transfection. A total of 15 µg of DNA was mixed with linear 25 kDa polyethyleneimine (PEI) (Polysciences, Eppenheim, Germany) and incubated for 20 min at room temperature, then the DNA/PEI mixture was added to plates. After 24 h from transfection, HCT 116-pCMV6-PIWIL1 cells were analyzed by immunofluorescence using rabbit anti-PIWIL1 (ab12337, Abcam, Cambridge, UK); fluorescence images were collected with a LEICA DM6000 B Confocal Microscope and used to evaluate and quantify transfection efficiency, which was found to be ~20%.